It is estimated that over 50% of human proteins are glycosylated. Many health and disease biomarkers are glycosylated proteins and specific glycoforms may be correlated with health or disease state. This includes glycosylated proteins linked to cancer, diabetes, inflammation and other medical conditions, as well as development. Furthermore, glycosylated proteins are not just limited to human or mammals, but are found in all eukaryotic systems, as well as prokaryotes, Archaea and plants.
Antibodies are glycoproteins. The glycans on an antibody can be structurally heterogeneous and can vary significantly in health and disease. During antibody production in vitro, the glycans attached to an antibody are affected not only by the cell type used for its production, but also by the cell culture conditions. Changes in nutrient availability, pH, cell density and CO2 levels can markedly alter the antibody glycoforms produced by the cells. For therapeutic antibodies, this can affect tissue distribution, serum half-life, resistance to proteolysis, complement activation, antibody-dependent cytotoxicity (ADCC), and inflammation. Consequently, it is desirable to manufacture therapeutic antibodies within specific regulatory-approved limits for glycoform variation.
Glycan profiling of glycoproteins such as antibodies requires methods for testing and identifying glycoforms in an antibody sample. Analysis of glycan structure is an unstandardized, time-consuming and low-throughput process with deglycosylation incubation times of as much as 16 hours under conditions that not only result in partial deglycosylation but also risk damage to the proteins. (Jenkins, et al., Mol Biotechnol. 39(2):113-8 (2008); Liu, et al., J Pharm Sci. 97(7):2426-47 (2008). Attempts to shorten the incubation period have resulted in the use of detergents that affect the protein integrity and hence functionality and interfere with mass spectrometry. Antibodies can be difficult to deglycosylate because glycans can be buried within the molecule's structural fold. Standard methods denature the antibody structure to expose the glycans using such methods as extreme heat and harsh denaturants like sodium dodecyl sulfate (SDS). However, these methods have several drawbacks: for example SDS can denature the enzymes used to remove glycans, SDS is difficult to remove, and even trace amounts of SDS can interfere with sample analysis methods like mass spectrometry. These methods are also time-consuming. Alternative methods attempt to increase the rate of deglycosylation by using a high concentration of Peptide-N-Glycosidase F (PNGase F). However, this approach does not overcome the presumed and undesirable bias associated with partial deglycosylation. Partial deglycosylation may result in certain glycoforms being preferentially released from the protein over others. This approach is also costly and is not readily scalable as it requires significant amounts of PNGase F.
Current approaches to improve deglycosylation for antibody characterization continue to result in ever more complex methods, require additional time to process, are inefficient, and incompletely remove glycans. Furthermore, the production of deglycosylated antibodies is currently limited to site-mutagenesis or bacterial expression systems. Unfortunately, these systems have difficulties in producing properly assembled and folded antibodies in any meaningful quantity.